Two Panels of Steroid Receptor Luciferase Reporter Cell Lines for Compound Profiling

Sedlák, David; Paguio, Aileen; Bartůněk, Petr

doi:10.2174/138620711795222446

Cited by 39 publications

(23 citation statements)

References 28 publications

(29 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This drug is unable to activate the MR in vitro 27 , in contrast to many clinically used steroids 9,10 . Fluticasone is actually a partial antagonist at the MR receptor 27 as well as a direct enhancer of potassium channel activity 32 ; however the latter effect occurs at concentrations orders of magnitude higher than GR activation.…”

Section: Discussionmentioning

confidence: 89%

“…Our characterization of steroids as GR selective or as dual MR/GR agonists relied on published in vitro studies using cell lines with reporter constructs, in which a reporter gene is placed downstream from regulatory binding sites to which the activated GR or MR transcription factors bind and activate transcription (transactivation) 9,10 . However, such experiments may not fully describe how a drug will act in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…However, recent in vitro studies show that many clinically used steroids (including e.g. 6-α methylprednisolone and triamcinolone) can also activate the mineralocorticoid receptor (MR) with similar potency 9,10 . The MR was originally viewed only as the target of aldosterone, promoting sodium reabsorption in the kidney.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Blocking the Mineralocorticoid Receptor Improves Effectiveness of Steroid Treatment for Low Back Pain in Rats

Xie²,

Strong³

et al. 2014

Anesthesiology

View full text Add to dashboard Cite

Background Localized inflammation of lumbar dorsal root ganglia (DRG) may contribute to low back pain. Local injections of corticosteroids used for low back pain are sometimes ineffective. Many corticosteroids activate not only the target glucocorticoid receptor (GR) but also the mineralocorticoid receptor (MR), which may have pro-inflammatory effects countering the effects of GR activation. Methods A low back pain model was implemented in rats (n = 6 -10 per group) by locally inflaming the L5 DRG. Sensory neuron excitability and mechanical hypersensitivity of the hind paws were measured. Tested steroids were applied locally to the inflamed DRG or orally. Results The selective MR blocker eplerenone reduced pain behaviors when given orally starting at the time of surgery, or starting 7 days later. The highly GR-selective agonist fluticasone, applied locally to the inflamed DRG, was much more effective in reducing mechanical hypersensitivity. The MR/GR agonist 6-α methylprednisolone, commonly injected for low back pain, reduced mechanical hypersensitivity when applied locally to the DRG, but was less effective than fluticasone. Its effectiveness was improved by combining it with local eplerenone. All tested steroids reduced hyperexcitability of myelinated sensory neurons (n = 71 – 220 cells per group) after inflammation, particularly abnormal spontaneous activity. Conclusions This preclinical study indicates the MR may play an important role in low back pain involving inflammation. Some MR effects may occur at the level of the sensory neuron. It may be useful to consider the action of clinically used steroids at the MR as well as at the GR.

show abstract

Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Blocking the Mineralocorticoid Receptor Improves Effectiveness of Steroid Treatment for Low Back Pain in Rats

Xie²,

Strong³

et al. 2014

Anesthesiology

View full text Add to dashboard Cite

show abstract

“…Sedlak et al (2011) identified some artifacts introduced by the Gal4/LBD reporter assays with the estrogen receptor (ER) in contrast to their full-length receptor reporter counterparts (Sedlak et al, 2011). However, it also may be true that the PXR-FL has an inherently reduced responsiveness in the reporter systems, related for example, to the presence of functional domains in the full protein not present in the LBD construct, or differences in stability.…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

et al. 2013

View full text Add to dashboard Cite

The pregnane X receptor (PXR) (nuclear receptor NR1I2) is a ligand activated transcription factor, mediating responses to diverse xenobiotic and endogenous chemicals. The properties of PXR in fish are not fully understood. Here we report on cloning and characterization of full-length PXR of zebrafish, Danio rerio, and pxr expression in vivo. Initial efforts gave a cDNA encoding a 430 amino acid protein identified as zebrafish pxr by phylogenetic and synteny analysis. The sequence of the cloned Pxr DNA binding domain was highly conserved, with 74% identity to human PXR, while the ligand-binding domain (LBD) of the cloned sequence was only 44% identical to human PXR-LBD. Sequence variation among clones in the initial effort prompted sequencing of multiple clones from a single fish. There were two prominent variants, one sequence with S183, Y218 and H383 and the other with I183, C218 and N383, which we designate as alleles pxr*1 (nr1i2*1) and pxr*2 (nr1i2*2), respectively. In COS-7 cells co-transfected with a PXR-responsive reporter gene, the full-length Pxr*1 (the more common variant) was activated by known PXR agonists clotrimazole and pregnenolone 16α-carbonitrile but to a lesser extent than the full-length human PXR. Activation of full-length Pxr*1 was only 10% of that with the Pxr*1 LBD. Quantitative real time PCR analysis showed prominent expression of pxr in liver and eye, as well as brain and intestine of adult zebrafish. The pxr was expressed in heart and kidney at levels similar to that in intestine. The expression of pxr in liver was weakly induced by ligands for mammalian PXR or CAR (NR1I3). The results establish a foundation for PXR studies in this vertebrate model. PXR allelic variation and the differences between the full-length PXR and the LBD in reporter assays have implications for assessing the action of PXR ligands in zebrafish.

show abstract

“…The cells used to develop these assays should have two specific requirements: a reporter system driven by an ARE and an abundant expression of AR. A variety of reporter genes have been used in the past years including β-lactamase [20], β-galactosidase [21–26] and luciferase [15, 18, 27–32] reporter genes. To develop an androgen reporter line superior to the ones available, we chose not to use yeast cells for several reasons.…”

Section: Discussionmentioning

confidence: 99%

Development of a novel cell based androgen screening model

Campana

Rege

Turcu

et al. 2016

The Journal of Steroid Biochemistry and Molecular Biology

View full text Add to dashboard Cite

The androgen receptor (AR) mediates the majority of androgen effects on target cells. The DNA cis-regulatory elements that respond to AR share sequence similarity with cis-regulatory elements for glucocorticoid, mineralocorticoid and progesterone receptors (GR, MR and PR respectively). As a result, many of the current AR screening models are complicated by inaccurate activation of reporters by one of these receptor pathways. Identification of more selective androgen testing systems would be beneficial for clinical, pharmacological and toxicologic screening of AR activators. The present study describes the development of a selective androgen-responsive reporter cell line that expresses AR but does not express GR, MR and PR. CV1 cells were stably transduced to express human AR and an androgen-responsive gaussia luciferase gene. Clonal populations of AR expressing cells were isolated. Quantitative RT-PCR (qPCR) and western analysis confirmed stable integration of AR in the most responsive clonal line which was named ‘CV1-ARluc’. Stimulation of CV1AR-luc with androgenic ligands (testosterone and 5α-dihydrotestosterone) for 18 h caused an increase in luciferase activity in a dose-dependent manner. Other steroid hormones including aldosterone, cortisol, and progesterone did not stimulate luciferase response. The CV1-ARluc also increased luciferase activity when treated with human serum extracts. In conclusion, the CV1-ARluc cells provide a novel model system for screening of new AR agonists and antagonists and can determine the androgenic activity of human serum samples.

show abstract

Two Panels of Steroid Receptor Luciferase Reporter Cell Lines for Compound Profiling

Cited by 39 publications

References 28 publications

Blocking the Mineralocorticoid Receptor Improves Effectiveness of Steroid Treatment for Low Back Pain in Rats

Blocking the Mineralocorticoid Receptor Improves Effectiveness of Steroid Treatment for Low Back Pain in Rats

Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

Development of a novel cell based androgen screening model

Contact Info

Product

Resources

About